1. Field of the Invention
The present invention relates to conjugates between siRNA (small interfering RNA) molecules and hydrophilic polymers, which can effectively be used for delivering an siRNA for treatment of cancers and other infectious diseases, and polyelectrolyte complex micelles formed by ionic interactions between the conjugates and multifunctional cationic compounds.
2. Description of the Related Art
Safe and effective gene transfer techniques for gene therapy have been studied for a long time, resulting in development of various gene transfer vehicles and gene delivery systems. In particular, vectors based on adenoviruses and retroviruses, and nonviral vectors using liposomes, cationic lipids and cationic polymers have been developed as gene transfer vehicles. However, there are significant problems when viruses are used as vehicles for transfer of therapeutic genes into target cells. There is no evidence that the transferred genes cannot lead to the malfunction of host genes and/or the activation of oncogenes after integration into the host chromosome. In addition, if viral genes are continuously expressed even at a small amount, autoimmune response can be induced. Moreover, if a variant of the virus used as a gene transfer vehicle emerges in a host, the host can become infected with the variant virus, and the host immune system cannot effectively protect itself from the variant virus. For these reasons, rather than the viral vectors, gene delivery systems using liposomes, cationic lipids, or polymers are preferred, and related studies are aiming to improve drawbacks of each system. Such nonviral gene transfer vectors are less effective than the viral vectors, but are advantageous in terms of safety due to their mild side effects and being economical due to low cost production, thereby allowing industrial production of improved nonviral vectors.
The most important emerging approach in drug delivery system and gene delivery system now is a target specific delivery. When a drug is administered directly in vivo, every organ and every cell of a human body are equally attacked by the drug, and this, desired or not, damages normal cells and tissues as well as damaged or infected ones. To prevent such a problem, a large amount of research into drug delivery systems (DDS) has been performed to develop a technique used for selective delivery of drugs and genes. For example, a typical tissue/cell specific ligand such as folate, galactose, antibody and the like can be introduced directly into a drug or be conjugated with a drug transporter, so that delivery efficiency can be maximized while side effects of the drug on normal cells can be minimized. The delivery efficiency in a cell is expected to be maximized by employing such tissue/cell specific ligands to the preparation of a transporter for developing a gene-based therapeutic agent.
Meanwhile, a micelle is spontaneously formed by self-assembly of molecules having both hydrophilic and hydrophobic moieties at a specific ratio in an aqueous environment to maximize thermodynamic stability. The inside of the micelles is hydrophobic and thus can easily entrap water-insoluble drugs, and the surface of the micelles is hydrophilic and thus the micelle system facilitates solubilization of the water-insoluble drugs, drug delivery carrier and so on. Micelles having the hydrophobic core and the hydrophilic shell are stabilized in an aqueous environment by hydrophobic interaction, or stabilization of micelles can be achieved by ionic interaction between polyelectrolytes having opposite charges. A polyethylene glycol (PEG)-conjugated polyelectrolyte spontaneously associates with another polyelectrolyte having an opposite charge to form complex having a micellar structure, which are called polyelectrolyte complex micelles (Kataoka, K., et al., Macromolecules 29:8556-8557 (1996)). The polyelectrolyte complex micelles are more attractive than other drug delivery systems, such as microspheres or nanoparticles, due to there properties of having a very small size and a very uniform size distribution, and being a self-associated structure, thereby facilitating quality control and reproduction of pharmaceutical preparations.
Polymers used for drug delivery to a body should be biocompatible. A representative example of such biocompatible polymers is PEG. PEG, which has been approved for in vivo use by the U.S. Food and Drug Administration (FDA), has been utilized for a long time in a broad range of applications from improvement of protein characteristics, surface modification of polymers, and gene delivery. PEG, which is one of the most widely used biocompatible polymers, has excellent water solubility, and low toxicity and immunogenicity. In addition, PEG can strongly inhibit absorption of proteins to the polymers used in drug delivery by modifying the surface properties of the polymers.
Meanwhile, siRNA is a substance having generated a lot of interest as a gene-based therapeutic agent ever since it has been reported to have an excellent inhibitory effect of the expression of a specific gene in a zooblast (animal cell). In effect, because of its high activity and precise gene selectivity, siRNA is expected to be an alternative therapeutic agent to an antisense oligonuceotide (ODN) currently being used as a therapeutic agent as a result of 20-years of research. The siRNA is a short, double-helix RNA strand which can suppress expression of a targeted mRNA having complementary base sequence to the siRNA.
siRNA has very low stability and is quickly degraded in vivo, thus its therapeutic efficiency deteriorates quickly. Even though the dose of expensive siRNA can be increased, the anionic nature of siRNA hinders it from permeating a cell membrane with negative charge, leading to low levels of siRNA transfer into intracellular compartments (Celia M. et al., Chemical and Engineering News December 22:32-36 (2003)). In addition, although siRNA is double stranded, the linkage of a ribose sugar in the RNA is chemically very unstable compared with that of a deoxyribose sugar in the DNA. Thus, the majority of siRNA has a half-life of about 30 minutes in vivo and is quickly degraded.
According to a recent study into the enhancement of stability of siRNA, various functional groups have been introduced into siRNA to protect the siRNA from lyases (Frank Czauderna et al., Nucleic Acids Research 31:2705-2716 (2003)). Nevertheless, the technology for securing the stability and effective cell membrane permeability of siRNA is still in development phases. The study also suggested that because of the instability of siRNA molecules in blood high-concentrations of siRNA should be continuously introduced in order to get therapeutic effects of the siRNA. Unfortunately however, this method is reported to be highly inefficient. Even from an economic aspect, there is a need to develop technology for a novel transporting agent that facilitates intracellular transfer of siRNA as a gene-based therapeutic agent.